Adenylate cyclase is a membrane-bound enzyme which plays a key role in the regulation of cellular function and the mediation of hormonal action. Although significant knowledge of the properties of this enzyme and its relationship to hormone receptors has been forthcoming, a clearcut understanding of its regulation, the mechanism of its linkage to the receptor in the membrane and the cofactors and lipid milieu critical for its activity, can only come following its purification and subsequent characterization. Previous attempts to purify this enzyme as has been the case with a variety of other membrane-bound proteins has met with little success. Since membrane proteins display a spectrum of hydrophobicity, we attempted to separate cyclase from more hydrophobic proteins utilizing a new class of non-charged resins consisting of long chain alkyl groups linked to agarose via ether bonds. Following such hydrophobic resolution, cyclase displayed a more typical hydrophilic character and detergent was no logner required to prevent aggregation. Affinity purification with nucleotide-Sepharose resins was now successful and initial experiments have indicated a 5,000-fold purification can be obtained. It is our plan to scale up these procedures and to achieve homogeneity. We also will attempt to raise a neutralizing antibody to the enzyme which should provide a powerful tool in mapping enzyme location, topology and mobility in the membrane. While inhibiting enzymatic activity with this antibody during maximal beta-receptor occupancy, one can also directly approach the question of whether beta-receptor mediated effects are mediated solely and universally through cyclic AMP generation. Finally, direct characterization of the purified preparation will be undertaken including determination of Stokes radius, partial specific volume, molecular weight and symmetry. Of significant interest will be attempts to probe its hydrophobic binding site and to obtain a picture of just how it is anchored to the membrane. Subunit composition, both in terms of physical structure and functional characteristics, will be defined hopefully providing insights into the enzyme's regulatory mechanisms.